WO1995033838A1 - Microorganisms permitting the intracellular polyhydroxy alkanoate synthesis with simultaneous extracellular polysaccharide synthesis and processes for producing the same - Google Patents

Abstract

So far, the commercialisation on a large scale of the environmentally harmless PHB and PHA biopolymers has failed because of the very high production costs involved in the production of these substances by biotechnological fermentation processes. The present invention allows these biopolymers to be produced at lower costs. The invention describes microorganisms which synthesize PHB or PHA intracellularly and permit the extracellular synthesis of at least one polysaccharide on account of the expression of at least one protein having the enzymatic activity of a hexosyltransferase. It also describes processes for preparing such microorganisms.

Description

Microorganisms permitting the intracellular polyhydroxy al anoate synthesis with simultaneous extracellular polysaccharide synthesis and processes for producing the same

The present invention relates to microorganisms which are capable of intracellularly synthesizing polyhydroxy alkanoate and of expressing extracellular enzymes which catalyze the synthesis of different polysaccharides whereby di-, oligo- and polysaccharides that are present in the culture medium are cleaved. These enzymes are in particular hexosyltransferases. Furthermore, the present invention relates to processes for preparing such microorganisms.

Nowadays, microorganisms are used on a large scale for the synthesis of different substances in biotechnological production processes. The synthesis of complex biopolymers, such as for instance polyhydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) , inter alia, is of great importance. These biopolymers are of particular commercial interest, because they possess thermoplastic properties which are comparable to those of synthetically prepared plastics, such as polypropylene.

The biopolymers PHA and PHB can, in part, replace conven¬ tional, industrially produced polymers. They are of potential interest for the packaging industry, as they possess several advantages compared to conventional plastics such as for instance 100% biodegradability and superior environmental compatibility and the advantage of allowing renewable resources to be used as starting materials for the production. Thus, they can contribute to a reduction of plastic waste difficult to recycle.

So far, it has not been possible to commercialize these biopolymers on a large scale, because of their high production costs, resulting inter alia from the great complexity of the production technology, the high costs for the production plants, the expensive processing of the products and the high prices of the raw materials. The production of synthetic petrochemical plastics, such as for instance polyproplylene is less expensive, with the result that these products are, as a rule, inexpensive and prepared in large quantities.

PHB is a polyester of D(-) -3-hydroxy butyric acid. The term polyhydroxy alkanoate (PHA) as used hereinafter comprises the polymers of 3-hydroxy butyric acid, polymers of related hydroxyalkanoates, such as 3-hydroxy valerate, 3-hydroxy hexanoate and 3-hydroxy decanoate, and moreover copolymers and mixtures of these hydroxy alkanoates.

So far, PHB and PHA have only been found in prokaryots and are used by many bacteria species as substances for the intracellular storage of carbon and energy. They are stored in the cells in granules and can amount to 90% of the dry weight of the cells.

At present, a copolymer of PHB and polyhydroxy valerate is prepared on an industrial scale by Imperial Chemical Industries PLC and marketed under the name of BIOPOL. In this production process, the microorganism Alcaligenes eutrophus is used (Byrom, 1992, FENS Microbiol. Rev. 103:247-250). Said microorganism is cultured in a glucose salt medium in a fed batch reactor under nutrient conditions permitting cell growth during the first 60 hours only and PHB synthesis during the subsequent 48 hour phase. This leads to a high PHB accumulation in the cells. In order to isolate the PHB from the cells, the latter are separated from the culture medium, and the PHB is, as a rule, extracted from the cells by means of a solvent ( ethanol; chloroform/methylene chloride) , and subsequently precipitated and dried in vacuo.

The excessive costs incurred in the production of PHA via Alcaligenes eutrophus are attributable in part to the small hydrocarbon substrate spectrum of this microorganism. The wild type strain only utilizes fructose as a sugar and the sugar acid gluconate (Wilde, 1962, Arch. Mikrobiol. 43:109- 137; Gottschalk et al., 1964, Arch. Mikrobiol. 48:95-108) . Mutants derived from the wild type strain can also grow on glucose (Schlegel and Gottschalk, 1965, Biochem. Z. 341:249- 259) . These strains are ^' preferably used for technical fermentation, because glucose is less expensive than fructose. Suitable glucose sources include disaccharides, such as sucrose (glucose-fructose) , maltose (glucose- glucose) or oligosaccharides, such as dextran or dextrin, some of which form during the processing of agricultural products as secondary or waste products.

However, the disadvantage in using these less expensive substrates is that the microorganisms used for the PHA production, including Alcaligenes eutrophus, are unable to import said di- or oligosaccharides, because of the absence of corresponding transport systems for the import into the cells. Hence, these substrates must be hydrolyzed prior to being used and must thus be converted to the corresponding hexose monomers. Again, such treatments are time-consuming and expensive.

In order to achieve a reduction in the PHA production costs by increasing the substrate spectrum of Alcaligenes eutrophus, attempts ^' have been made on the one hand to produce strains through mutagenesis which are able to use other less expensive substrates than does the wild type, and on the other hand, to introduce heterologous genes into the microorganism Alcaligenes eutrophus which allow alternative substrates, such as sucrose to be transported into the cells. However, both approaches failed. Mutagenesis requires a very large number of clones to be screened for a suitable clone to be obtained. The introduction of heterologous genes coding for transport systems of alternative substrates often involves the difficulty that several genes constitute such transport systems. Therefore, all essential genes must be transferred and provided with promoters that are recognized in the host to be used. The difficulty frequently encountered here is that the coordinated expression of these genes in the new host does not proceed properly and therefore an effective expression of the transport system does not occur. Therefore, in order to transfer transportation systems for alternative substrates to another microorganism, it is, as a rule, necessary to know the precise control of the expression of the genes.

In spite of the numerous efforts made, it has not yet been possible to broaden the spectrum of substrates for PHA producing microorganisms to include inexpensive substrates contributing to a lowering of the PHA production costs. There continues to be an urgent demand for the development of economic PHB and PHA production methods permitting a commercialization of these biopolymers on a larger scale.

Hence, the present invention addresses the problem of providing microorganisms and processes permitting a less expensive PHA production in microorganisms.

The object is achieved by the embodiments characterized in the patent claims.

The present invention thus relates to microorganisms intracellularly producing PHB or PHA and permitting the extracellular synthesis of at least one polysaccharide on account of the expression of at least one extracellular protein having the enzymatic activity of a hexosyl- transferase.

The microorganisms of the invention are preferably microorganisms belonging to the genus Alcaligenes, in particular Alcaligenes eutrophus or the microorganism E. coli. which is capable of intracellularly synthesizing PHA on account of the introduction of genes encoding enzymes for the PHA synthesis.

Moreover, the invention relates to processes for preparing such microorganisms wherein DNA sequences coding for at least one extracellular protein having the enzymatic activity of a hexosyltransferase are introduced into a microorganism which synthesizes PHB or PHA intracellularly. Said DNA sequences are linked to regulatory DNA sequences for controlling the transcription and contain a signal sequence ensuring the secretion of the synthesized proteins.

The invention relates in particular to such a process comprising the following steps:

(a) construction of an expression cassette ensuring the expression of an extracellular protein possessing hexosyltransferase activity;

(b) transformation of a microorganism with the expression cassette constructed in step (a) ; and

(c) culturing the transformed microorganism under conditions permitting the expression of said protein.

In principle, any microorganism capable of synthesizing PHB or PHA intracellularly may used as the abovementioned microorganism; bacteria belonging to the genus Alcaligenes and bacteria of the species E. coli, incorporating DNA sequences encoding enzymes for the PHA synthesis are preferred. An expression cassette as indicated in process step (a) which permits the expression of an extracellular protein possessing hexosyltransferase activity as a rule contains the following DNA sequences:

(i) a promoter sequence ensuring the expression of a downstream DNA sequence in the host organism that has been selected;

(ii) a DNA sequence encoding at least one protein possessing hexosyltransferase activity and being linked to the promoter in sense orientation; and

(iii) a DNA sequence at the 3' end of the DNA sequence mentioned in (ii) which serves as the termination signal for transcription.

Such an expression cassette is preferably located on a vector molecule which apart from the expression cassette contains the following DNA sequences:

(v) one or more selection marker gene(s) ; and (vi) DNA sequences ensuring the replication of the vector DNA in the microorganism selected, if the vector DNA is not integrated . into the genome of the microorganis .

It is not always absolutely necessary for individual sequences to be present, depending on the system used for expression. For instance the DNA sequence mentioned in item (iii) above and the selection marker gene mentioned in item (v) is not necessary in all cases. Furthermore, the DNA sequences, permitting the expression of a protein possessing hexosyltransferase activity do not have to be located on a vector in each case, but may be integrated into the genome of the host organism via a homologous or non-homologous recombination in one or more copies at one or more loci. In this case the DNA sequence mentioned in item (vi) does not have to be present. Moreover, it is possible that not only one but several DNA sequences coding for hexosyltransferases of different types are expressed in an organism.

The construction of such expression cassettes and vectors and the manipulation of the DNA sequences used may be carried out according to conventional techniques known to the expert. Such techniques are described in detail for instance in Sambrook et al., 1989, second edition, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York; Methods in Enzymology, 1979, 1983, 1986 and 1987, Academic Press, New York, Vols. 68, 100, 101, 118 and 152-155; and DNA-Cloning, 1985 and 1987, volumes I, II, III, Glover, editors, IRL Press, Oxford.

The DNA sequence naturally controlling the transcription of the hexosyltransferase gene selected can be used as the promoter sequence, if it is active in the organism selected. However, this sequence may also be exchanged for other promoter sequences. It is possible to use promoters which cause a constitutive expression of the gene as well as inducible promoters allowing a downstream DNA sequence to be controlled by external factors. Bacterial and viral promoter sequences possessing these properties have been described in the literature in detail. Promoters permitting a particularly strong expression of downstream DNA sequences are for instance the T7 promoter (Studier et al., 1990, in Methods in Enzymology 185:60-89), lacuvS, trp-lacUV5 (DeBoer et al, in Rodriguez, R.L. and Chamberlin, M.J. , (Eds.), Promoters, Structure and Function; Praeger, New York, 1982, pp. 462-481; DeBoer et al, 1983, Proc. Natl. Acad. Sci. USA 80:21-25), lp_lf rac (Boros et al., 1986, Gene 42:97-100) or the ompF-promoter. Known promoters include those which are inducible by sucrose, for instance from Bacillus amyloliquefaciens. The DNA sequence mentioned in item (ii) coding for a protein with the enzymatic activity of a hexosyltransferase may have different origins. Such enzymes and DNA sequences encoding them are for instance known from different microorganisms. The enzymes or DNA sequences encoding them described in the following are used according to a preferred embodiment of the invention.

In the present invention the term hexosyltransferases is to mean enzymes catalysing reactions whose mechanism is distinguished by the fact that a hexose is directly transferred from a di-, oligo- or polysaccharide to an acceptor, which as a rule is a growing polysaccharide chain. In this case, catalysis requires neither activated glucose derivatives, such as occurring in the polysaccharide synthesis in plants and animals, nor cofactors. The energy necessary for the polymerization of the hexose residues is directly obtained through the cleavage of the glycosidic bond in the corresponding di-, oligo- or polysaccharide.

The hexosyltransferases using sucrose as a substrate are differentiated on the basis of whether they transfer the glucose residue (glucosyltransferases) or the fructose residue (fructosyltransferases) from the sucrose molecule to a growing polysaccharide chain. The reaction products formed are fructose and glucans in the first case and glucose and fructans in the second case.

Glucosyltransferases using sucrose as the substrate generally catalyze reactions of the following type:

sucrose + (glucan)_n > fructose + (glucan)_n+1

Depending on the way in which the glucose molecules are linked to each other in the glucan and on the occurrence of branchings, different glucans may occur as reaction products. Extracellular glucosyltransferases from Streptococcus species which catalyze the synthesis of glucans possessing different properties are known. These enzymes are divided into three groups:

(a) glucosyltransferases which synthesize water-soluble glucans, the majority of which is α-1,6 linked, the so- called dextrans (GTF S-type)

(b) glucosyltransferases, which synthesize water-insoluble glucans, the majority of which is α-1,3 linked, the so- called mutans (GTF I-type)

(c) glucosyltransferases, which synthesize a combination of water soluble and water insoluble glucans (GTF-SI type) .

Genes encoding glucosyltransferases of the S-type, I-type and Si-type have already been isolated from four different Streptococcus species (for an overview see Giffard et al., 1993, J. Gen. Microbiol. 139:1511-1522) .

Moreover, a gene has been described coding for a dextransucrase (sucrose: 1, 6-α-D-glucane 6-α-D-glucosyltrans- ferase, E.C. 2.4.1.5.) from Leuconostoc mesenteroides (WO 89/12386) . This transferase is likewise a glucosyl¬ transferase which uses sucrose as a substrate. The resulting glucan, i.e. dextran, consists predominantly of α-1,6 linked glucose molecules, with the parallel chains being cross- linked among each other.

DNA sequences coding for dextranmaltases or dextran dextrinaεes may also be used.

Another glycosyltransferase which uses sucrose as the substrate is the amylosucrase (also designated: sucrose: 1,4- α-D-glucan 4-α-glucosyltransferase, E.C. 2.4.1.4.) . This enzyme catalyses the reaction: sucrose + (α-1 , 4-D-glucan) _n —> fructose + (α-l,4-D- glucan) _n+1

So far, amylosucrase has been found only in a few bacteria species, mainly including Neisseria species (MacKenzie et al., 1978, Can. J. Microbiol. 24:357-362). A DNA sequence containing a region coding for amylosucrase activity has been isolated from a genomic DNA library of Neisseria polyεaccharea. Said DNA sequence is contained in the plasmid pNB2 (DSM 9196) .

Fructosyltransferases using sucrose as the substrate have also been described. They catalyze reactions of the following type:

sucrose + (fructose)_ —> glucose + (fructose)_n+1

The products produced in this reaction are fructans, apart from glucose. They contain one sucrose molecule to which fructose polymers are added and which acts as a starter molecule of the polymerization reaction. Depending on the type of linkage of the fructose molecules, the synthesized fructans can be divided into two groups:

(a) (2 —>1) linked β-D-fructans (inulin type)

(b) (2 —>6) linked β-D-fructans (phlein or levan type) .

In conformity with the two different types of fructans which can occur as synthesis products, the fructosyltransferases are likewise divided into two types which are known by the common names of levansucrase (sucrose:β-D-fructosy1- transferase, E.C. 2.4.1.10.) and inulosucrase (E.C. 2.4.1.9.), respectively.

DNA sequences coding for levansucrases and inulosucrases, respectively, have so far been isolated from different microorganisms. They include DNA sequences from Bacillus a yloliquefaciens (Tang et al, 1990, Gene 96:89-93) , Bacillus subtilis (Stein etz et al, 1985, Mol. Gen. Genetics 200: 220-228 and Erwinia amylovora (Geier and Geider, 1993, Phys. Mol. Plant Pathology 42:387-404; DE 42 27 061.8 and WO 94/04692) . They code for levansucrases which catalyze the synthesis of polyfructans of the levan type. Moreover, a DNA sequence coding for a fructosyltransferase from Streptococcus mutans has been described (Shiroza et al., 1988, J. Bacteriology 170:810-816; Sato and Kura itsu, 1986, Infect. Immun. 52:166-170) . This enzyme synthesizes a fructan of the inulin type.

In addition to hexosyltransferases which use sucrose as the substrate, hexosyltransferases using maltose aε the substrate are known. For instance, an amylomaltase (also designated: α-1,4-glucan:D-glucose 4-glucosyltransferase, E.C. 2.4.1.3.) from Escherichia coli has been described for which the following reaction mechanism has been proposed:

maltose + (1,4-α-glucan)_n —> D-glucose + (1,4-α-glucan)_n+1

(Palmer et al., 1976, Eur. J. Biochem. 69:105-115; Haselbarth et al., 1971, Biochim. Biophyε. Acta 227:2996- 3012) . The gene coding for this cytosolic enzyme participating in the maltose metabolism of E. coli, has likewise been described (Pugsley and Dubreuil, 1988, Mol. Microbiol. 2:473-479).

If the DNA sequence coding for a protein possessing hexosyl¬ transferase activity, which has been selected for use, does not possess in its 5 ' region a DNA sequence coding for a signal peptide sequence ensuring the secretion of the hexosyltransferase, then a DNA sequence coding for such a signal peptide sequence can be inserted between the promoter and the coding DNA sequence. The sequence to be used must, in each case, be in the same reading frame as the DNA sequence coding for the enzyme. Such signal peptide sequences are for instance found in the gene coding for levansucrase from bacteria of the Bacillus genus (Borchert and Nagarajan, 1991, J. Bacteriol. 173:276-282) .

On the one hand, the process according to the invention allows microorganisms that are employed for the PHA production by fermentation processes to be cultured using inexpensive substrates. The hexosyltransferases secreted into the medium lead to the cleavage of suitable di-, oligo- or polysaccharides present in the medium. This cleavage results in the release of hexoses which are imported by the microorganisms and can be used for the cell growth or the synthesis of intracellular products.

Preferred embodiments of the microorganisms and the process of the invention are those embodiments in which the hexosyl¬ transferases use disaccharides, in particular sucrose or maltose as substrates. The use of sucrose as a substrate in the culturing medium and expresεion of a secreted glucoεyltranεferaεe causes fructose which can be imported by the microorganismε to be released in the medium. As described in Linko et al. (1993, Appl. Microbiol. Biotech. 39:11-15), fructose iε a particularly suitable substrate for the intracellular PHA synthesis and compared to other carbon sources leads to an especially high PHA portion of the dry weight of the cells. The process of the invention therefore also provides the possibility of producing the advantageous, though relatively expensive substrate fructose from the considerably less expensive subεtrate sucrose and of lowering the costs.

Moreover, the secreted hexosyltransferases enable the extracellular synthesis of different polysaccharides as has been described above. The majority of theεe polyεaccharides are of considerable commercial importance. There iε a variety of possible uses of dextran for instance in the food sector, pharmaceutical sector, e.g. as blood plasma substitutes or for increasing the viscosity of aqueous solutionε, and in the chemical industry, e.g. as bases for dextran gels.

The α-1,4 glucans formed by amylosucrase are of particular commercial interest, aε their chemical εtructure corresponds to the amylose portion of plant εtarch. Amyloεe is widely used inter alia in the food, paper and textile industries and in the production of cyclodextrinε. Starch itεelf, which so far has been the sole source for obtaining α-1,4 glucans, however, consistε of two components. Apart from the amylose which iε an unbranched chain of α-1,4 linked glucose units, starch contains another component, the amylopectin. Thiε is a highly branched polymer of glucose units, which, apart from the α-1,4 links, shows branches of the glucoεe chainε through α-1,6 linkε. On account of the different structure of these two components and the physico-chemical properties resulting therefrom, the two components also offer quite different possibilities of use. In order to benefit from the individual components directly, it is necesεary to obtain them in pure form. Both components can be obtained from starch, which, however, requires several purification steps and is time consuming and involves costs.

In spite of the many efforts made, it has not yet been possible to prepare pure amylose by means of microorganisms or in plants. The production of pure amylose by means of biotechnological processes for providing a chemically uniform basic substance for the different industrial purposes of use is therefore of εpecial interest.

Moreover, polyfructose iε an inexpenεive fructoεe εource, because it is stable, non-hygroscopic, and therefore possesses good storage properties. Given its viscoεity properties, polyfructose would alεo εeem to be a suitable thickening agent. In this connection it is also of importance that fructose can only insufficiently (i.e by microorganisms) be utilized, and therefore polyfructose is ideally suited as an additive to low calorie foodstuffs. Moreover, polyfructose is suitable for encapsulating flavours, colorants and other additives, as it cannot absorb water and therefore permits storage under atmospheric conditions.

Furthermore, polyfructose is also of interest as a replacement for chemically produced linear polymers that are biologically not degradable.

The α-1,4 glucans which are syntheεized by amylomaltase and which under εuitable conditionε can achieve chain lengthε similar to those of amylose, are used for corresponding purpoεes as has already been described above for the glucans synthesized by amylosucraεe.

The extracellularly εyntheεized polysaccharideε can be iεolated directly from the culture medium. The intracellularly formed polyhydroxy alkanoates can be isolated from the cells after separation of the cells from the culture medium. Therefore, the simultaneous extracellular synthesis of these polysaccharides in conjunction with the intracellular PHA synthesiε can entail a further reduction of the production costs and can thus contribute to an increaεe in the rentability of the whole PHA production process.

On the one hand, the construction of a vector comprising an expresεion cassette for the expression of an extracellular hexosyltransferase can be carried out in such a way that the expression cassette which is composed of the following elements

(i) control elements for initiating the transcription (promoter) (ii) a DNA sequence coding for a protein with hexoεyl- tranεferaεe activity and being linked to the promoter in sense orientation, and ₍iii) a DNA sequence at the 3' end of the DNA sequence specified in (ii) above, serving as a termination signal for transcription

and enables the expression of a translatable RNA, is constructed in a vector suitable for the host chosen for use.

On the other hand, the expression casεette can be constructed in a conventional cloning vector, isolated from the cloning vector with the uεe of εuitable reεtriction enzymeε and inεerted into a vector suitable for the transformation of the host selected for use.

Moreover, an expression vector can be prepared by inserting a DNA sequence coding for a protein posεesεing hexosyl- tranεferaεe activity into a vector already containing control elements for the initiation of the transcription and a DNA sequence εerving aε a termination εignal for transcription. In this case, a single restriction site or a polylinker into which the DNA sequence to be expressed may be inserted lies between the control elements for the initiation of the transcription and the termination signal.

There exist a great number of cloning vectors which are useful for preparing the DNA sequences mentioned in the proceεs steps and which contain a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples of such vectors are pBlueskript plaεmids, pBR322, pUC-series, M13mp-series, pACYC184 etc. The desired sequence may be inserted into the vector at a suitable restriction site. The reεulting plaεmid iε used for the tranεformation of E. coli cells. Aε a rule, the transformation can be carried out according to εtandard methods as described in Sambrook et at. (Molecular Cloning; A Laboratory Manual, 1989, second edition, Cold Spring Harbor Laboratory Presε N.Y.). The tranεfor ed E. coli cells are cultured in a εuitable medium, harveεted and subjected to lysiε. The plaεmid is then isolated. In general, the methods for the characterization of the resulting plasmid DNA are restriction analyεis, gel electrophoresis, εequencing reactionε and other methods used in biochemistry and molecular biology. After each operation, the plasmid DNA can be cleaved with restriction endonucleases and the DNA fragments that are isolated can be linked to other DNA sequences.

Where Alcaligenes eutrophus is used for the expresεion of an extracellular hexosyltransferase, vectors, so-called "broad host range" vectors are available, with the aid of which a plurality of gram negative bacteria can be transformed. These contain DNA sequences which ensure replication of the plasmid DNA both in the bacteria such as E. coli and in Alcaligenes eutrophus. Theεe vectorε include for instance the plasmids pLAFR3 and pLAFR6 (Staskawicz et al. , 1987, J. Bacteriol. 169:5789-5794; Bonas et al., 1989, Mol. Gen. Genet. 218:127-136) .

The introduction of "broad hoεt range" vectors into Alcaligenes eutrophus is carried out by conjugation with an E. coli strain containing the corresponding plasmid and by conjugation with a helper strain by means of triparental mating (Figurski and Helinski, 1979, Proc. Natl. Acad. Sci. USA 76:1648-1652; Ditta et al, 1980, Proc. Natl. Acad. Sci. USA 77:7347-7351) .

The transformed microorganism is cultured in media which must meet the requirements of the particular hoεt, in particular conεidering the pH value, temperature, ventilation etc.

If a culture medium is used that contains sucrose or maltose, the polysaccharideε of the corresponding type are synthesized in the medium with the si ultaneouε release of hexoses. The hexoεes can be imported by the microorganiεm that is cultivated. The synthesized polysaccharides can be isolated from the culture medium after termination of the fermentation procesε. The isolation of the intracellularly synthesized PHB from the cells after recovery of the latter is carried out according to methods known in the literature.

The invention also relates to the microorganisms produced by the process of the invention.

Another embodiment of the present invention relates to the uεe of DNA sequences coding for a protein possessing the enzymatic activity of a hexosyltranεferase, for preparing microorganismε which synthesize PHB or PHA intracellularly and which, on account of the expression of at leaεt one protein poεεeεεing the enzymatic activity of a hexosyl¬ transferase permit the extracellular synthesis of at least one polysaccharide in the culture medium.

Moreover, the invention relates to the use of microorganisms prepared according to one of the processes of the present invention, for combining an intracellular PHB or PHA synthesis with a simultaneous extracellular synthesiε of a polyεaccharide on account of the εecretion of a protein having the enzymatic activity of a hexosyltransferase.

Another embodiment of the invention relates to processes for preparing PHB, PHA or a polysaccharide using a microorganism of the invention.

Abbreviations used

GTF glucosyltransferase

PHB polyhydroxybutyrate Figure 1 shows colonies of A. eutrophus, which contain the plasmid pGE9 and were cultured on 0.2% sucrose-containing FN-medium at 28°C for 2 days. After exposure to iodine vapour for about 5 minutes the colonieε are surrounded by a blue-coloured halo.

Figure 2A shows a microscopic picture of a heat-fixed preparation of A. eutrophus transconjugantε containing the pGE9 plaε id and stained with 1% (w/v) of Nile Blue A solution (1000 fold enlargement)

Figure 2B shows a microscopic picture as deεcribed in Figure 2A, prepared using light with a wave length of 460 nm. At this wave length the Nile Blue A dye which binds to PHB grana shows fluorescence (1000 fold enlargement) .

The invention iε illustrated by the exampleε which, as will be appreciated, do not to limit the scope of the invention in any way.

Example 1

Construction of the "broad-host" vector pGE9 for the expression of an extracellular amylosucrase

The construction of a "broad host" vector permitting the expression of an extracellular amylosucrase in Alcaligeneε eutrophuε was carried out uεing a genomic DNA fragment from Neisseria polysaccharea containing the coding region for an amylosucrase. Such a fragment waε iεolated as the PstI fragment from the vector pNB2 (DSM 9196) . Said fragment comprises the DNA sequence depicted in SeqID No. 1. The fragment was ligated into the vector pGE151 linearized by PstI (a derivative of vector pKM9-6, Kortlύcke et al. J. Bacteriol. 174 (1992), 6277-6289) . The resulting vector pGE9 was transformed into the Escherichia coli strain S17-1 (Simon et al., Bio/Technology 1 (1983), 784-791) and plated onto selection medium (YT-medium containing 15 μg/ml of tetracyclin) .

Example 2

Conjugative plasmid tranεfer between E. coli and Alcaligenes eutrophus H16 (Friedrich et al. , J. Bacteriol. 147 (1981) 198-205)

The donor strain (E. coli S17-1 with pGE9) prepared according to Example 1 was incubated in 4 ml YT-medium at 37"C overnight. The recipient (A. eutrophus H16; Wilde, Arch. Mikrobiol. 43 (1962) , 109-13 ) was likewise incubated in 4 ml of YT-medium at 28^ overnight.

The overnight cultures were pelleted, waεhed with YT-medium once and then pelleted again. The pelletε were placed in 10 fold concentration into a physiological saline εolution (0.9% of NaCl solution). 0.2 ml each of the donor and recipient concentrates were mixed and plated onto solid YT- medium. This batch was incubated for about 6 hours at 28°C without being disturbed.

The plate was then washed with 2 ml of physiological saline solution. 10^"1, 10~² and 10^-3 dilutionε of the cell εuspension were prepared. 200 μl of each dilution were plated onto a εelection medium (FN-medium containing 15 μg/ml of tetracyclin) and incubated at 28°C until colonies had grown (after approximately 2 days) . Negative controls were obtained by plating the overnight cultures onto FN- medium with tetracyclin and incubating them at 28°C also. Example 3

Examination of the transconjugans for amylosucrase activity and simultaneous production of poly-β-hydroxbutyrate (PHB)

Some of the colonies prepared according to example 2 and grown on the selection medium were transferred to FN-medium containing tetracyclin and 0.2% sucrose in addition, and were incubated at 28°C for 2 days. The transconjugantε were expoεed to iodine vapour in order to demonstrate glucans formed by the amyloεucrase. In the case of colonies with secreted amylosucrase, the exposure to iodine vapor resultε in the formation of a blue-coloured halo in conεequence of the formation of linear α-1,4 glucans (see Figure 1) .

For the production of PHB, the transconjugants were placed into a defined minimal medium (MM) which additionally contained 1% of εucroεe and were incubated at 28°C for four days (Peoples et al. J. Biol. Chem. 264 (1989), 15298- 15303) .

From these cultures, heat fixed preparations were prepared to be examined under the microscope. These preparations were stained with 1% (w/v) of Nile blue A solution according to the method of Oεtle et al. (Appl. Environ. Microbiol. 44 (1982) , 238-241) (εee Figure 2A) . Thiε colorant bindε to PHB grana and fluoreεceε an orange light at a stimulation wavelength of 460 nm (εee Figure 2B) .

The presence of α-1,4 glucans was shown by staining the cell-free εupernatant with Lugol εolution. Solutions and media used

Lugol solution: 13.1 mM of I₂ 39.6 mM of KI dissolved in H-O (distilled twice)

YT-medium: 0.8% of bacto-trypton 0.5% of yeast extract 0.5% of NaCl

FN-medium: 0.02% of MgS0₄ ^• 7H₂0

0.001% of CaCl₂ ' ' 2H₂0

0.5 ^* 10^"3% of FeCl₃ " 6H₂0

(stock solution dissolved in

0.1 N HC1)

0.2% of NH₄C1

0.2% of fructose

+ 1 ml SL6-solution

+ 100 ml 10 x H16 buffer ad 1000 ml with H₂0 (diεtilled twice)

lOx H16 buffer 59.2 g/1 Na₂HP0₄ 15 g/1 KH₂P0₄

SL6-solution 0. .1 g/i ZnS0₄ ' 7H₂0

0. .03 g/i MnS0₄ ^• 4H₂0

0. .2 g/i CoCl₂ ' 6H₂0

0. .01 g/i CuS0₄ ^• 2H₂0

0. .02 g/i NiCl₂ ^•6H₂0

0. .03 g/i Na₂MoO 4 ^{" 2H}2° Trace element solution 20 τng/1 CuS0₄ 5H₂0 100 mg/1 ZnS0₄ 6H₂0 100 mg/1 MnS0₄ 4H₂0 2.6 g/1 CaCl₂ 2H₂0

MM: 0.39 g/1 of MgS0₄

0.45 g/1 of K₂S0₄

12 ml/1 1.1 M H₃P0₄

15 mg/1 FeS0₄ ' 7H₂0

24 ml/1 trace element solution the pH value is adjusted with

NaOH to 6.8 after sterilization, NH₄Cl is added up to a final concentration of 0.05% and fructoεe up to a final concentration of 1% (w/v) .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Institut fuer Genbiologische Forschung Berlin

GmbH

(B) STREET: Ihnestrasse 63

(C) CITY: Berlin

(E) COUNTRY: DE

(F) POSTAL CODE (ZIP) : 14195

(G) TELEPHONE: +49 30 8300070 (H) TELEFAX: +49 30 83000736

(ii) TITLE OF INVENTION: Microorganisms permitting the intracellular polyhydroxy alkanoate synthesis with simultaneous extracellular polysaccharide synthesis and processes for producing the same

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: DE P 44 20 223.7

(B) FILING DATE: 06-JUN-1994

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2883 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Neisseria polysaccharea

(vii) IMMEDIATE SOURCE:

(A) LIBRARY: genomic library in pBluescriptll SK

(B) CLONE: pNB2

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION:939..2780 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GAGTTTTGCG TTCCCGAACC GAACGTGATG CTTGAGCCGA ACACCTGTCG GCAAGCGCTG 60

ACCGCCTTTT GCCCCATCGA CATCGTAACA ATCGGTTTGG TGGCAAGCTC TTTCGCTTTG 120

AGCGTGGCAG AAAGCAAAGT CAGCACGTCT TCCGGCCTTT CGGCATCACC GCAATTTTGC 180

AGATGTCCGC GCCGCAGTCC TCCATCTGTT TCAGACGGCA TACGATTTCT TCTTGCGGCG 240

GCGTGCGGTG AAACTCATGA TTGCAGAGCA GGGCGATGCC GTTTTTTTGA GCATGCCACG 300

GCGCCGGAGC GGTTTCGCCG GAAAAAAGCT CGATATCGAT AATGTCGGGC AGGCGGCTTT 360

CAATCAGCGA GTCGAGCAGT TCAAAATAAT AATCGTCCGA ACACGGGAAC GAGCCGCCTT 420

CGCCATGCCG TCTGAACGTA AACAGCAGCG GCTTGTCGGG CAGCGCGTCG CGGACGGTCT 480

GCGTGTGGCG CAATACTTCG CCGATGCTGC CCGCGCATTC CAAAAAATCG GCGCGGAACT 540

CGACGATATC GAAGGGCAGG TTTTTGATTT GGTCAAGTAC GGCGGAAAGT ACGGCGGCAT 600

CGCGGGCGAC AAGCGGCACG GCGATTTTGG TGCGTCCGCT TCCGATAACG GTGTTTTTGA 660

CGGTCAGGGC TGGTGTGCAT GGCGGTTGTT GCGGCTGAAA GGAACGGTAA AGACGCAATT 720

ATAGCAAAGG CACAGGCAAT GTTTCAGACG GCATTTCTGT GCGGCCGGCT TGATATGAAT 780

CAAGCAGCAT CCGCATATCG GAATGCAGAC TTGGCACAAG CCTGTCTTTT CTAGTCAGTC 840

CGCAGTTCTT GCAGTATGAT TGCACGACAC GCCCTACACG GCATTTGCAG GATACGGCGG 900

CAGACCGCGT CGGAAACTTC AGAATCGGAG CAGGCATC ATG TTG ACC CCC ACG 953

Met Leu Thr Pro Thr 1 5

CAG CAA GTC GGT TTG ATT TTA CAG TAC CTC AAA ACA CGC ATC TTG GAC 1001 Gin Gin Val Gly Leu lie Leu Gin Tyr Leu Lys Thr Arg lie Leu Asp 10 15 20

ATC TAC ACG CCC GAA CAG CGC GCC GGC ATC GAA AAA TCC GAA GAC TGG 1049 lie Tyr Thr Pro Glu Gin Arg Ala Gly lie Glu Lys Ser Glu Asp Trp 25 30 35

CGG CAG TTT TCG CGC CGC ATG GAT ACG CAT TTC CCC AAA CTG ATG AAC 1097 Arg Gin Phe Ser Arg Arg Met Asp Thr His Phe Pro Lys Leu Met Asn 40 45 50

GAA CTC GAC AGC GTG TAC GGC AAC AAC GAA GCC CTG CTG CCT ATG CTG 1145 Glu Leu Asp Ser Val Tyr Gly Asn Asn Glu Ala Leu Leu Pro Met Leu 55 60 65

GAA ATG CTG CTG GCG CAG GCA TGG CAA AGC TAT TCC CAA CGC AAC TCA 1193 Glu Met Leu Leu Ala Gin Ala Trp Gin Ser Tyr Ser Gin Arg Asn Ser 70 75 80 85 TCC TTA AAA GAT ATC GAT ATC GCG CGC GAA AAC AAC CCC GAT TGG ATT 1241 Ser Leu Lys Asp He Asp He Ala Arg Glu Asn Asn Pro Asp Trp He 90 95 100

TTG TCC AAC AAA CAA GTC GGC GGC GTG TGC TAC GTT GAT TTG TTT GCC 1289 Leu Ser Asn Lys Gin Val Gly Gly Val Cys Tyr Val Asp Leu Phe Ala 105 110 115

GGC GAT TTG AAG GGC TTG AAA GAT AAA ATT CCT TAT TTT CAA GAG CTT 1337 Gly Asp Leu Lys Gly Leu Lys Asp Lys He Pro Tyr Phe Gin Glu Leu 120 125 130

GGT TTG ACT TAT CTG CAC CTG ATG CCG CTG TTT AAA TGC CCT GAA GGC 1385 Gly Leu Thr Tyr Leu His Leu Met Pro Leu Phe Lys Cys Pro Glu Gly 135 140 145

AAA AGC GAC GGC GGC TAT GCG GTC AGC ACG TAC CGC GAT GTC AAT CCG 1433 Lys Ser Asp Gly Gly Tyr Ala Val Ser Thr Tyr Arg Asp Val Asn Pro 150 155 160 165

GCA CTG GGC ACA ATA GGC GAC TTG CGC GAA GTC ATT GCT GCG CTG CAC 1481 Ala Leu Gly Thr He Gly Asp Leu Arg Glu Val He Ala Ala Leu His 170 175 180

GAA TCG CAT TTC CGC CGT CGT CGA TTT TAT CTT CAA CCA CAC CTC CAA 1529 Glu Ser His Phe Arg Arg Arg Arg Phe Tyr Leu Gin Pro His Leu Gin 185 190 195

CGA ACA CGA ATG GCG CAA CGC TGC GCC GGC GAC CCG CTT TTC GAC AAT 1577 Arg Thr Arg Met Ala Gin Arg Cys Ala Gly Asp Pro Leu Phe -Asp Asn 200 205 210

TTC TAC TAT ATT TTC CCC GAC CGC CGG ATG CCC GAC CAA TAC GAC CGC 1625 Phe Tyr Tyr He Phe Pro Asp Arg Arg Met Pro Asp Gin Tyr Asp Arg 215 220 225

ACC CTG CGC GAA ATC TTC CCC GAC CAG CAC CCG GGC GGC TTC TCG CAA 1673 Thr Leu Arg Glu He Phe Pro Asp Gin His Pro Gly Gly Phe Ser Gin 230 235 240 245

CTG GAA GAC GGA CGC TGG GTG TGG ACG ACC TTC AAT TCC TTC CAA TGG 1721 Leu Glu Asp Gly Arg Trp Val Trp Thr Thr Phe Asn Ser Phe Gin Trp 250 255 260

GAC TTG AAT TAC AGC AAC CCG TGG GTA TTC GCG CAA TGG CGG GCG AAA 1769 Asp Leu Asn Tyr Ser Asn Pro Trp Val Phe Ala Gin Trp Arg Ala Lys 265 270 275

TGC TGT TCC TTG CCA ACT TGG GCG TTG ACA TCC TGC GTA TGG ATG CGG 1817 Cys Cys Ser Leu Pro Thr Trp Ala Leu Thr Ser Cys Val Trp Met Arg 280 285 290

TTG CCT TTA TTT GGA AAC AAA TGG GGA CAA GCT GCG AAA ACC TGC GCA 1865 Leu Pro Leu Phe Gly Asn Lys Trp Gly Gin Ala Ala Lys Thr Cys Ala 295 300 305 GCG CAC GCC CTC ATC CGC GCG TTC AAT GCC GTT ATG CGT ATT GCC GCG 1913 Ala His Ala Leu He Arg Ala Phe Asn Ala Val Met Arg He Ala Ala 310 315 320 325

CCC GCC GTG TTC TTC AAA TCC GAA GCC ATC GTC CAC CCC GAC CAA GTC 1961 Pro Ala Val Phe Phe Lys Ser Glu Ala He Val His Pro Asp Gin Val 330 335 340

GTC CAA TAC ATC GGG CAG GAC GAA TGC CAA ATC GGT TAC AAC CCC CTG 2009 Val Gin Tyr He Gly Gin Asp Glu Cys Gin He Gly Tyr Asn Pro Leu 345 350 355

CAA ATG GCA TTG TTG TGG AAC ACC CTT GCC ACG CGC GAA GTC AAC CTG 2057 Gin Met Ala Leu Leu Trp Asn Thr Leu Ala Thr Arg Glu Val Asn Leu 360 365 370

CTC CAT CAG GCG CTG ACC TAC CGC CAC AAC CTG CCC GAG CAT ACC GCC 2105 Leu His Gin Ala Leu Thr Tyr Arg His Asn Leu Pro Glu His Thr Ala 375 380 385

TGG GTC AAC TAC GTC CGC AGC CAC GAC GAC ATC GGC TGG ACG TTT GCC 2153 Trp Val Asn Tyr Val Arg Ser His Asp Asp He Gly Trp Thr Phe Ala 390 395 400 405

GAT GAA GAC GCG GCA TAT CTG GGC ATA AGC GGC TAC GAC CAC CGC CAA 2201 Asp Glu Asp Ala Ala Tyr Leu Gly He Ser Gly Tyr Asp His Arg Gin 410 415 420

TTC CTC AAC CGC TTC TTC GTC AAC CGT TTC GAC GGC ACG TTC GCT CGT 2249 Phe Leu Asn Arg Phe Phe Val Asn Arg Phe Asp Gly Thr Phe Ala Arg 425 430 435

GGC GTA CCG TTC CAA TAC AAC CCA AGC ACA GGC GAC TGC CGT GTC AGT 2297 Gly Val Pro Phe Gin Tyr Asn Pro Ser Thr Gly Asp Cys Arg Val Ser 440 445 450

GGT ACA GCC GCG GCA TTG GTC GGC TTG GCG CAA GAC GAT CCC CAC GCC 2345 Gly Thr Ala Ala Ala Leu Val Gly Leu Ala Gin Asp Asp Pro His Ala 455 460 465

GTT GAC CGC ATC AAA CTC TTG TAC AGC ATT GCT TTG AGT ACC GGC GGT 2393 Val Asp Arg He Lys Leu Leu Tyr Ser He Ala Leu Ser Thr Gly Gly 470 475 480 485

CTG CCG CTG ATT TAC CTA GGC GAC GAA GTG GGT ACG CTC AAT GAC GAC 2441 Leu Pro Leu He Tyr Leu Gly Asp Glu Val Gly Thr Leu Asn Asp Asp 490 495 500

GAC TGG TGC CAA GCA GCA ATA AGA GCG ACG ACA GCC GTT GGG CCA CCG 2489 Asp Trp Cys Gin Ala Ala He Arg Ala Thr Thr Ala Val Gly Pro Pro 505 510 515

TCC GCG CTA CAA CGA AGC CCT GTA CGC GCA ACC GAA CGA TCC GTC GAC 2537 Ser Ala Leu Gin Arg Ser Pro Val Arg Ala Thr Glu Arg Ser Val Asp 520 525 530 CGC AGC CGG CAA ATC TAT CAG GGC TTG CGC CAT ATG ATT GCC GTC CGC 2585 Arg Ser Arg Gin He Tyr Gin Gly Leu Arg His Met He Ala Val Arg 535 540 545

CAA AGC AAT CCG CGC TTC GAC GGC GGC AGG CTG GTT ACA TTC AAC ACC 2633 Gin Ser Asn Pro Arg Phe Asp Gly Gly Arg Leu Val Thr Phe Asn Thr 550 555 560 565

AAC AAC AAG CAC ATC ATC GGC TAC ATC GCA ACA ATG CGC TTT TGG CAT 2681 Asn Asn Lys His He He Gly Tyr He Ala Thr Met Arg Phe Trp His 570 575 580

TCG GTA ACT TCA GCG AAT ATC CGC AAA CCG TTA CCG CGC ATA CCC TGC 2729 Ser Val Thr Ser Ala Asn He Arg Lys Pro Leu Pro Arg He Pro Cys 585 590 595

AAG CCA TGC CCT TCA AGG CGC ACG ACC TCA TCG GTG GCA AAA CTG TCA 2777 Lys Pro Cys Pro Ser Arg Arg Thr Thr Ser Ser Val Ala Lys Leu Ser 600 605 610

GCC TGAATCAGGA TTTGACGCTT CAGCCCTATC AGGTCATGTG GCTCGAAATC 2830

Ala

GCCTGACGCA CGCTTCCCAA ATGCCGTCTG AACCGTTTCA GACGGCATTT GCG 2883

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 614 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Leu Thr Pro Thr Gin Gin Val Gly Leu He Leu Gin Tyr Leu Lys 1 5 10 15

Thr Arg He Leu Asp He Tyr Thr Pro Glu Gin Arg Ala Gly He Glu 20 25 30

Lys Ser Glu Asp Trp Arg Gin Phe Ser Arg Arg Met Asp Thr His Phe 35 40 45

Pro Lys Leu Met Asn Glu Leu Asp Ser Val Tyr Gly Asn Asn Glu Ala 50 55 60

Leu Leu Pro Met Leu Glu Met Leu Leu Ala Gin Ala Trp Gin Ser Tyr 65 70 75 80

Ser Gin Arg Asn Ser Ser Leu Lys Asp He Asp He Ala Arg Glu Asn 85 90 95

Asn Pro Asp Trp He Leu Ser Asn Lys Gin Val Gly Gly Val Cys Tyr 100 105 110 Val Asp Leu Phe Ala Gly Asp Leu Lys Gly Leu Lys Asp Lys He Pro 115 120 125

Tyr Phe Gin Glu Leu Gly Leu Thr Tyr Leu His Leu Met Pro Leu Phe 130 135 140

Lys Cys Pro Glu Gly Lys Ser Asp Gly Gly Tyr Ala Val Ser Thr Tyr 145 150 155 160

Arg Asp Val Asn Pro Ala Leu Gly Thr He Gly Asp Leu Arg Glu Val 165 170 175

He Ala Ala Leu His Glu Ser His Phe Arg Arg Arg Arg Phe Tyr Leu 180 185 190

Gin Pro His Leu Gin Arg Thr Arg Met Ala Gin Arg Cys Ala Gly Asp 195 200 205

Pro Leu Phe Asp Asn Phe Tyr Tyr He Phe Pro Asp Arg Arg Met Pro 210 215 220

Asp Gin Tyr Asp Arg Thr Leu Arg Glu He Phe Pro Asp Gin His Pro 225 230 235 240

Gly Gly Phe Ser Gin Leu Glu Asp Gly Arg Trp Val Trp Thr Thr Phe 245 250 255

Asn Ser Phe Gin Trp Asp Leu Asn Tyr Ser Asn Pro Trp Val Phe Ala 260 265 270

Gin Trp Arg Ala Lys Cys Cys Ser Leu Pro Thr Trp Ala Leu Thr Ser 275 280 285

Cys Val Trp Met Arg Leu Pro Leu Phe Gly Asn Lys Trp Gly Gin Ala 290 295 300

Ala Lys Thr Cys Ala Ala His Ala Leu He Arg Ala Phe Asn Ala Val 305 310 315 320

Met Arg He Ala Ala Pro Ala Val Phe Phe Lys Ser Glu Ala He Val 325 330 335

His Pro Asp Gin Val Val Gin Tyr He Gly Gin Asp Glu Cys Gin He 340 345 350

Gly Tyr Asn Pro Leu Gin Met Ala Leu Leu Trp Asn Thr Leu Ala Thr 355 360 365

Arg Glu Val Asn Leu Leu His Gin Ala Leu Thr Tyr Arg His Asn Leu 370 375 380

Pro Glu His Thr Ala Trp Val Asn Tyr Val Arg Ser His Asp Asp He 385 390 395 400

Gly Trp Thr Phe Ala Asp Glu Asp Ala Ala Tyr Leu Gly He Ser Gly 405 410 415 Tyr Asp His Arg Gin Phe Leu Asn Arg Phe Phe Val Asn Arg Phe Asp 420 425 430

Gly Thr Phe Ala Arg Gly Val Pro Phe Gin Tyr Asn Pro Ser Thr Gly 435 440 445

Asp Cys Arg Val Ser Gly Thr Ala Ala Ala Leu Val Gly Leu Ala Gin 450 455 460

Asp Asp Pro His Ala Val Asp Arg He Lys Leu Leu Tyr Ser He Ala 465 470 475 480

Leu Ser Thr Gly Gly Leu Pro Leu He Tyr Leu Gly Asp Glu Val Gly 485 490 495

Thr Leu Asn Asp Asp Asp Trp Cys Gin Ala Ala He Arg Ala Thr Thr 500 505 510

Ala Val Gly Pro Pro Ser Ala Leu Gin Arg Ser Pro Val Arg Ala Thr 515 520 525

Glu Arg Ser Val Asp Arg Ser Arg Gin He Tyr Gin Gly Leu Arg His 530 535 540

Met He Ala Val Arg Gin Ser Asn Pro Arg Phe Asp Gly Gly Arg Leu 545 550 555 560

Val Thr Phe Asn Thr Asn Asn Lys His He He Gly Tyr He Ala Thr ^'565 570 575

Met Arg Phe Trp His Ser Val Thr Ser Ala Asn He Arg Lys Pro Leu 580 585 590

Pro Arg He Pro Cys Lys Pro Cys Pro Ser Arg Arg Thr Thr Ser Ser 595 600 605

Val Ala Lys Leu Ser Ala 610

Claims

Patent Claims

1. A microorganism which synthesizeε PHB or PHA intracellularly and permits the extracellular syntheεis of at least one polysaccharide, on account of the expreεsion of at least one protein having the enzymatic activity of a hexosyltransferase.

2. The microorganism according to claim 1, wherein the protein with the enzymatic activity of a hexosyl- tranεferaεe iε a glucoεyltranεferaεe or a fructoεyl- transferase.

3. The microorganism according to claim 2, wherein the glucosyltransferaεe iε an amylosucrase (EC 2.4.1.4), an amylomaltase (EC 2.4.1.3), a dextransucrase (EC 2.4.1.5), a glucosyltransferase of the S, I or SI type from a Streptococcus species, or a dextran dextrinase.

4. The microorganism according to claim 2, wherein the fructosyltranεferase is a levanεucraεe (EC 2.4.1.10) or an inuloεucrase (EC 2.4.1.9) .

5. The microorganism according to any one of claims 1 to 4, the microorganiεm belonging to the genus Alcaligenes or the species Escherichia coli.

6. A process for preparing a microorganism which synthesizes PHB or PHA intracellularly and permits the extracellular synthesis of at least one polyεaccharide,' wherein DNA εequenceε coding for at least one extracellular protein having the enzymatic activity of a hexosyltransferase are introduced into a micro¬ organism which εynthesizeε PHB or PHA intracellularly.

7. The proceεε according to claim 6, comprising the following εtepε:

(a) conεtruction of an expreεεion cassette ensuring the expresεion of an extracellular protein possessing hexosyltransferase activity;

(b) transformation of a microorganism with the expression cassette constructed in step (a) ; and

(c) culturing the transformed microorganism under conditions permitting the expreεεion of said protein.

8. The proceεs according to claim 6 or 7, wherein the protein possessing the enzymatic activity of a hexosyl- transferase is a glucosyltransferaεe or 'a fructosyltransferase.

9. The process according to claim 8, wherein the glucosyl- transferase is an amylosucraεe (EC 2.4.1.4), an amylo altase (EC 2.4.1.3), a dextransucrase (EC 2.4.1.5), a glucosyltransferase of the S, I or SI type from a Streptococcus species or a dextran dextrinase.

10. The procesε according to claim 8, wherein the fructosyltransferase is a levansucrase (EC 2.4.1.10) or an inulosucrase (EC 2.4.1.9) .

11. The process according to any one of claims 6 to 10, wherein the microorganism belongs to the genus Alcaligenes or the species Escherichia coli.

12. The use of DNA sequenceε coding for a protein having the enzymatic activity of a hexoεyltranεferase, for the preparation of microorganismε which εyntheεize PHB or PHA intracellularly and permit the extracellular synthesis of at least one polysaccharide on account of the expression of at least one extracellular hexosyl¬ transferase.

13. The use of a microorganism according to any one of claims 1 to 5 for preparing PHB, PHA or a polysaccharide.

14. A procesε for preparing PHB, PHA or a polysaccharide, wherein a microorganism according to any one of claims 1 to 5 is used.